Antibiotic resistance is a growing threat to human health, necessitating the development of new and effective therapeutics. Natural products have proven to be an important resource of biologically active small molecules, but new methods to hasten their discovery are clearly needed. A deeper understanding of natural product biosynthesis will aid natural product discovery efforts and potentially lead to new strategies for combinatorial synthesis of natural product-based compound libraries. Recent studies in the Clardy laboratory have uncovered several natural products, including andrimid, pantocin C, and the dapdiamides, that are encoded by gene clusters lacking the traditional condensation enzymes of non-ribosomal polypeptide synthetases and polyketide syntheses. Instead, these natural products appear to be constructed by non-canonical condensation enzymes with homology to catalysts from primary metabolism. This surprising discovery implies that entire classes of biosynthetic gene clusters may be misannotated as belonging to primary rather than secondary metabolism. Detailed study of these new bond-forming catalysts and their synthetic logic represents an exciting opportunity, potentially leading to the discovery of entirely new classes of natural products through sequence-based searching. In this project, I will focus on one non-canonical condensation catalyst, the enzyme DdaF from dapdiamide biosynthesis in Pantoea agglomerans. DdaF is intriguing not only for its bond-forming activity, but also because it demonstrates unusual substrate promiscuity that could allow for the production of libraries of compound analogues. I propose three specific aims. Aim 1: To elucidate the role of DdaF in dapdiamide biosynthesis by determining whether it is responsible for the first or the second bond-forming step. Aim 2: To investigate the substrate promiscuity of DdaF as a means of characterizing its potential utility for combinatorial biosynthesis. I will characterize the extent of DdaF's substrate tolerance by screening a library of substrate analogues designed to probe the structural requirements for acceptance by DdaF. Aim 3: To investigate the substrate promiscuity of DdaF by solving crystal structures with bound reactants and/or products. The structure of DdaF will serve as the basis for a comparative structural analysis with homologous enzymes from primary metabolism and for structure-based searching for novel DdaF homologues